This project concerns how cells regulate intracellular membrane trafficking, including the formation of intracellular vesicles by budding from larger intracellular membrane compartments, movement of vesicles from one region of the cell to another, and the fusion of intracellular membrane compartments with one another. Rab GTPases play a multitude of roles in vesicular transport, including regulation of vesicle formation, vesicle motility, vesicle tethering, membrane fusion and membrane remodeling. Rab GDP dissociation inhibitor (GDI) is a protein that binds to rabs, keeping rab in the GDP-bound state, as well as delivering and placing rabs in the correct membrane compartment. The mechanism for releasing rab from GDI and delivering the rab to the correct membrane is unknown and is likely to involve proteins that bind and interact with GDI. Dr. Cheney's laboratory and collaborators have previously cloned the Drosophila GDI gene, determined the Drosophila GDI crystal structure, isolated and characterized Drosophila GDI mutations and located the GDI mutations within the GDI crystal structure (Ricard et al., 2001). Prior work supported by NSF (#9874626, "RUI: Proteins that control interaction of rab GTPases and GDIls") resulted in the discovery of candidate proteins that interact with GDI and potentially could regulate the release of rab from GDI. The research that will be conducted under this award will extend this project by further characterizing the interacting proteins and investigating the nature of their interaction with GDI and role in rab delivery. The prior work has isolated a 30 kD protein that bound to mutant, but not wild-type, GDI in a GST-GDI pulldown assay. This protein could be GDI displacement factor (GDF) or a rab. The prior work has also discovered 14 genomic regions that interact genetically with a GDI null allele. These regions include Myosin V, Myosin VIIa, rab26, Mp20/Calponin, and commissureless. Finally, this prior work has isolated 6 mutations that interact with GDI. The aims of the current project are:
1, to identify the 30 kD protein and determine whether it triggers rab release from GDI to membranes; 2, to find the candidate gene in ech of 4 interacting genomic regions and determine if the candidate gene product binds to GDI, affecting rab release from GDI; and 3, to clone the genes for one mutation that interacts with GDI and determine if the protein encoded by this gene binds directly to GDI, affecting in rab release.
This research will investigate proteins that interact with GDI and will shed light on how rab is released from GDI and delivered correctly to membrane compartments. Further, this project will integrally and actively involve undergraduates in an exciting area of research.
